Studies of protein-folding mechanisms and partially folded states of proteins can lead to a deeper understanding of many human disease states. Because the folding process is often very complex, traditional ensemble methods are unable to resolve several key features of its dynamics. We propose to develop single-molecule fluorescence methods for the study of detailed conformational distributions and dynamics during multi-step protein folding reactions. Methods to monitor short-range distance changes in proteins, and for the non-perturbative immobilization of proteins will be developed. Further development and optimization of single-molecule FRET and fluorescence lifetime data acquisition and analysis methods will also be carried out. These and related single molecule methods will be applied to study the multi-step folding reaction of barnase. Structural information about the different folding states and their interconversion will be obtained using a combination of single-molecule FRET and short-range quenching. Using protein engineering, the influence of mutations on the observed single-molecule distributions and folding landscapes will be examined, and long-standing issues such as connectivity between states and cooperativity of transitions between them will be clarified. The influence of molecular crowding and chaperones on this folding reaction will be studied, providing insights into in-vivo mechanisms of protein folding. Finally, the interplay between barnase folding and binding to barstar will be studied. It is anticipated that the developed methods will be useful in the study disease-related proteins. [unreadable] [unreadable]